The U1 snRNP, in collaboration with other splicing factors, promotes juxtaposition of intron termini and nucleates formation of a catalytically active spliceosome poised to join a particular pair of exons. The long-term goal of the proposed research is to provide a molecular description of the RNA-RNA, RNA-protein, and protein-protein interactions required for these events by exploiting the facile genetic manipulations available in the fission yeast Schizosaccharomyces pombe. The first major goal will be to illuminate dynamic snRNA-substrate interactions that precede the first transesterification reaction through three avenues of investigation: i) To determine whether U1 and U5 snRNAs interact during the transition from commitment complex to catalytically active spliceosome, their pairing interactions with the splicing substrate will be modulated, singly and in combination; ii) To determine which snRNAs play decisive roles in activation of a non-consensus 5' splice site, compensatory base analysis will be performed; and iii) To identify factors that potentiate or destabilize U1 pairing to the 5' and 3' splice sites, conditional alleles will be used for suppressor selection and synergistic lethal screening; characterization of the mutations in the proteins and RNAs that emerge will provide information about how these components physically and/or functionally interact with U1. The second major goal will be to perform structure/function analysis and analyze components that interact with two factors known to mediate early events in premessenger RNA splicing using mutational analysis followed by suppressor selection: i) the large subunit of the heterodimeric splicing factor U2AF, which binds to the polypyrimidine tract in early ATP-independent complexes that commit an intron to splicing and subsequently promotes stable U2 snRNP binding to the branchpoint and ii) Sap49, a component of the multimeric splicing factor SF3b, which is believed to play a role in delivering and tethering the U2 snRNP to the branchpoint. The results of these experiments will facilitate the construction of detailed models for early events in premessenger RNA splicing. Because S. pombe contains pre-mRNAs with multiple introns bonded by sub-optimal splicing signals, the data generated should be particularly relevant to understanding both constitutive and alternative splicing in multicellular organisms.